Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 43

2. 2.5 % (w/v) Glutaraldehyde (GA) in 0.1 M sodium cacodylate
buffer pH 7.0. GA is commercially available as 8, 25, and 50 %
(w/v) solutions. Alternatively a fi xative containing a mixture
of PFA and GA may be employed ( see Note 2 ). GA fi xatives
should be freshly prepared, stored at 4 °C and used within 1
month.
3. Ethanol: aqueous solutions 30-100 % (v/v) for sample
dehydration.
4. LR White resin, hard grade, containing 0.5 % (w/v) benzoin
methyl ether catalyst (Agar Scientifi c).
5. Gelatin capsules (Agar Scientifi c).
6. Vectabond (Vector Laboratories, Peterborough, UK)-coated
Multitest 8-well glass slides (MP Biomedicals, Solon, USA).
3. Nickel grids (Agar Scientifi c) for electron microscopy.
4. In-house prepared glass knives for trimming blocks.
5. A diamond knife: a Histo Diamond knife for semi-thin sections
and/or an Ultra Diamond knife for ultrathin sections
(Diatome, PA, USA). Alternatively the highest quality in-house
prepared glass knives may be fi tted with a boat made from
waterproof tape and sealed with wax.

2.4 Immuno-
microscopy

1. Super PAP hydrophobic pen (Agar Scientifi c) for outlining
each well on the slide.
2. Phosphate-buffered saline (PBS): prepare a 10× stock with
1.37 M NaCl, 27 mM KCl, 100 mM Na 2 HPO 4 , 18 mM
KH 2 PO 4 (pH 7.4) and autoclave before storage. Alternatively,
use prepared 10× PBS (Severn Biotech, Kidderminster, UK).
Prepare a working solution by dilution of one part stock solu-
tion with nine parts water.
3. Blocking/antibody dilution buffers: PBS with 3 % (w/v) milk
protein (e.g., Marvel, Premier Beverages, UK) (PBS/MP) or
3 % (w/v) bovine serum albumin (Sigma-Aldrich) in PBS
(PBS/BSA).
4. Secondary antibodies: anti-rat immunoglobulin (IgG) (whole
molecule) reagents coupled to FITC and gold; mouse anti-
His; anti-mouse IgG coupled to FITC (Sigma-Aldrich), anti-
His coupled to AlexaFluor 488 (Serotec, Kidlington, UK);
anti-rat coupled to AlexaFluor 488 (Invitrogen).
5. Anti-fade reagent: Citifl uor glycerol/PBS AF1 (Agar Scientifi c).
6. Microscope slide cover slips.
7. Calcofl uor White: 0.25 % (w/v) in PBS (Fluorescent
Brightener 28, Sigma-Aldrich).
8. Toluidine Blue O: 0.1 % (w/v) in 0.1 M sodium phosphate
buffer pH 5.5.